DESCRIPTION

CD34 is a 105to 120-kDa transmembrane phosphoglycoprotein consisting of a cytoplasmic tail that contains PDZ-domain binding motifs, a single transmembrane helix and an extracellular domain that is heavily sialylated, O-linked glycosylated, and contains some N-linked glycosylation sites. It is expressed by the majority of hematopoietic stem/progenitor cells and also by a multitude of other nonhematopoietic cell types including muscle satellite cells, corneal keratocytes, interstitial cells, epithelial progenitors, and vascular endothelial cells. CD34 is widely used as a marker of vascular endothelial cells and hematopoietic stem/progenitor cells and mediates cell adhesion and lymphocyte homing by binding L-selectin and E-selectin ligands.

PRINCIPLE OF THE ASSAY

An anti-tag antibody is pre-coated onto the bottom of wells. After adding antigen or samples, Tagged-Capture antibody and HRP-conjugated detection antibody, a sandwich complex is formed in the solution. TMB acts as a HRP substrate, and the solution color will change from colorless to blue. A stop solution containing sulfuric acid turns the solution yellow. The color intensity is proportional to the quantity of bound protein, which is measurable at 450 nm with the correction wavelength set at 630 nm.

PRECISION

Intra-assay Precision (Precision within an assay) Three samples of known concentration were tested 8 times on one plate to assess intra-assay precision. Inter-assay Precision (Precision between assays) Three samples of known concentration were tested in 16 separate assays to assess inter-assay precision.

Intra-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	8	1,008.0	26.1	2.6
2	8	273.2	8.3	3.0
3	8	143.5	5.7	4.0

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	986.6	33.7	3.4
2	16	264.3	11.7	4.4
3	16	138.9	7.0	5.0

RECOVERY

The recovery of mouse CD34 spiked to three different levels throughout the range of the assay in lysate was evaluated.

Sample Type		Average% of Expected	Range (%)
Lysate	1:1,280	99	92-107
	1:2,560	105	100-110

LINEARITY

To assess the linearity of the assay, samples were diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay. (The cell lysate was initially diluted 1:160.)

		Lysate
1:2	Average% of Expected	100
	Range (%)	-
1:4	Average% of Expected	101
	Range (%)	96-105
1:8	Average% of Expected	102
	Range (%)	95-110
1:16	Average% of Expected	119
	Range (%)	117-121

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant mouse CD34 produced at Sunlong Medical™.